Spirochetes of the genus Borrelia, the causative agents of vector-borne Lyme borreliosis (LB) and relapsing fever (RF), display throughout their lifecycle a variety of abundant lipoproteins with distinct biological functions. Irrespective of the regulatory mechanisms governing the expression of different lipoproteins in different environments, the successful deployment of these spirochetal virulence factors hinges on (i) an efficient lipoprotein modification and transport system, and (ii) an accurate lipoprotein sorting machinery. The overall objective of this proposal is to gain key insights into these two important underlying aspects of spirochetal pathogenesis using the LB spirochete Borrelia burgdorferi as a model. Our preliminary studies indicate that Borrelia lipoproteins sorting signals localize to an N-terminal sequence of about ten residues after the N-terminal cysteine, yet differ from the ones characterized in E. coli. We have also begun to characterize B. burgdorferi BB0346 as a functional homolog of the periplasmic lipoprotein carrier LolA in E. coli. Protein homologs for molecular events in this pathway upstream, but not downstream of BB0346 were identified as well. We therefore hypothesize that (i) the Borrelia lipoprotein export machinery is similar to the one described in E. coli, but (ii) pathways at the outer membrane and (iii) the lipoprotein sorting rules diverge significantly from the ones described in other diderm bacteria. To test these hypotheses, we have formulated the following three specific aims: 1. To further define borrelial lipoprotein sorting signals by studying subcellular (mis)localization of fluorescent reporter proteins and lipoprotein mutants. 2. To further characterize the biological function of B. burgdorferi Lol protein homologs using co- immunoprecipitation, affinity purification, and complementation experiments. 3. To identify and characterize other components of the borrelial lipoprotein export machinery using novel mutagenic and proteomic approaches. These studies will (i) significantly increase our understanding of spirochetal virulence, (ii) shed more light on the evolution of bacterial protein export mechanisms in general, and (iii) may yield important clues for the design of future intervention strategies.